This proposal to examine the molecular basis of cholecystokinin (CCK) receptor structure, function, and regulation supports the long-term goal of understanding the roles of this gastrointestinal hormone in health and disease. CCK is important for nutrient assimilation, with roles in regulating post-cibal pancreatic secretion, gallbladder emptying, gut transit, and satiety. The general underlying hypothesis is that understanding the CCK receptor requires insights into its global structure and molecular interactions throughout its dynamic life. Component aims are directed to each of three key CCK receptor domains, (i) the external face that is responsible for ligand binding, (ii) the internal face that is important for signal initiation and receptor regulation, and (iii) the intramembranous face that represents a potential site for interactions with other membrane proteins. Each aim is driven by hypotheses dealing with current concepts of functions and interactions that might occur at that site, with relevance for mechanisms of pharmacoregulation. Aim 1 examines the hypothesis that ligands with distinct chemical structures can induce similar global receptor conformations by interacting with distinct receptor domains. Studies utilize photoaffinity labeling, receptor mutagenesis, and fluorescence transfer techniques to gain insights into the binding of peptide and non-peptidyl agonists and antagonists. Aim 2 explores the hypothesis that receptor phosphorylation exposes previously hidden receptor domains that can interact with regulatory molecules. Studies utilize over- expression competition strategies to identify important cytosolic domains, and use biochemical and molecular biological techniques to identify molecules interacting with these. Aim 3 explores intramembranous interactions with the receptor that could have functional significance. Bioluminescence resonance energy transfer and cross-linking techniques are utilized. The proposed work builds on the unique strengths and experience of the laboratory and the tools that have been developed and extensively characterized in previous cycles of this grant. Together, these studies should provide the finest level of molecular detail yet available for the structure, mechanism of binding, and mechanisms of regulation of any peptide hormone receptor in this superfamily, while providing insights useful for the development and refinement of receptor- and cell-specific therapeutic intervention.